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. 1999 Jul;104(2):137-46.
doi: 10.1172/JCI6093.

Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts

H Takayanagi  1 , T Juji, T Miyazaki, H Iizuka, T Takahashi, M Isshiki, M Okada, Y Tanaka, Y Koshihara, H Oda, T Kurokawa, K Nakamura, S Tanaka
Affiliations

Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts

H Takayanagi et al. J Clin Invest. 1999 Jul.

Abstract

Rheumatoid arthritis (RA) is characterized by a chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages, and plasma cells, all of which manifest signs of activation. Recent studies have revealed the essential role of osteoclasts in joint destruction in RA. Src family tyrosine kinases are implicated in various intracellular signaling pathways, including mitogenic response to growth factors in fibroblasts, activation of lymphocytes, and osteoclastic bone resorption. Therefore, inhibiting Src activity can be a good therapeutic strategy to prevent joint inflammation and destruction in RA. We constructed an adenovirus vector carrying the csk gene, which negatively regulates Src family tyrosine kinases. Csk overexpression in cultured rheumatoid synoviocytes remarkably suppressed Src kinase activity and reduced their proliferation rate and IL-6 production. Bone-resorbing activity of osteoclasts was strongly inhibited by Csk overexpression. Furthermore, local injection of the virus into rat ankle joints with adjuvant arthritis not only ameliorated inflammation but suppressed bone destruction. In conclusion, adenovirus-mediated direct transfer of the csk gene is useful in repressing bone destruction and inflammatory reactions, suggesting the involvement of Src family tyrosine kinases in arthritic joint breakdown and demonstrating the feasibility of intervention in the kinases for gene therapy in RA. off

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Figures

Figure 1
Figure 1
Efficiency of adenovirus-mediated gene transduction into human rheumatoid synovial cells in culture. The percentage of synoviocytes positive for β-gal expression increased in an moi-dependent manner. Data are expressed as mean ± SD of quintuple measurements.
Figure 2
Figure 2
Csk expression and the kinase activity of synoviocytes. (a) Western blot analysis shows that Csk virus induced Csk expression in human synovial cells in an moi-dependent manner without significant changes in expression of c-Src protein. Fold increase in Csk expression normalized by β-actin is shown below each blot. The results shown are from a single experiment typical of 3 experiments yielding identical results. (b) Immunofluorescence staining using anti-Csk antibody shows that WT virus–infected synoviocytes expressed a very small amount of Csk protein. (c) Diffuse cytoplasmic staining in Csk virus–infected (moi = 100) synoviocytes. (d) In vitro kinase assay, using enolase as a substrate demonstrated that kinase activity of c-Src, Fyn, and c-Yes was remarkably repressed in Csk-overexpressing synoviocytes, but the expression of each protein was unaffected by Csk expression. Relative kinase activity of each kinase is shown below. The results were derived from 3 separate experiments. Suppression on each kinase was statistically significant (*P < 0.05 Csk+ vs. Csk–).
Figure 3
Figure 3
Inhibitory effects of adenovirus-mediated Csk overexpression on cell growth of rheumatoid synoviocytes. (a) Cell growth curves of rheumatoid synoviocytes inoculated with WT, LacZ, or Csk virus (moi = 100). Proliferation of Csk virus–infected cells was strongly suppressed. Data represent mean ± SD of triplicate measurements (*P < 0.05 Csk vs. WT, Csk vs. LacZ). (b) Proportion of BrdU-positive cells in synoviocytes inoculated with WT, LacZ, or Csk virus. Percentage of BrdU-positive cells inoculated with Csk virus decreased in an moi-dependent manner, but WT or LacZ virus had no effect on cell growth rate. Data are expressed as mean ± SD of quintuple measurements (*P < 0.01 Csk vs. WT, Csk vs. LacZ).
Figure 4
Figure 4
Effect of adenovirus-mediated Csk overexpression on IL-6 production. (a) IL-6 concentration in conditioned medium of rheumatoid synoviocytes inoculated with WT, LacZ, or Csk virus detected by ELISA. IL-6 production was reduced to about 50% below the normal level in rheumatoid synoviocytes inoculated with Csk virus at an moi of 10 or greater. Data are expressed as mean ± SD derived from 3 separate experiments (*P < 0.05 vs. LacZ). (b) IL-6 mRNA detected by Northern blot analysis in WT, LacZ, or Csk virus–infected synoviocytes. Significant decrease in IL-6 mRNA signals was observed in synovial cells infected with Csk virus at an moi of 10 or greater. Fold expression of IL-6 mRNA normalized by β-actin is shown below each blot. Data are expressed as the mean derived from 3 separate experiments (*P < 0.05 vs. LacZ).
Figure 5
Figure 5
Effect of adenovirus-mediated Csk on bone-resorbing activity of osteoclasts. (a) Active resorption pit formation by WT virus–infected osteoclasts after removal of adherent cells. Arrowheads indicate 2 major pits surrounded by several other pits. (b) No resorption pit was seen when inoculated with Csk virus (moi = 100). (c) Relative resorbed area was drastically suppressed by Csk virus in an moi-dependent manner. Data are expressed as mean ± SD of triplicate measurements (*P < 0.05 vs. LacZ).
Figure 6
Figure 6
In vivo expression of transgenes by adenovirus vectors. (a) In situ β-gal staining of the ankle synovial membrane, dissected 7 days after injection of LacZ virus, revealed a diffuse β-gal expression in the synoviocytes of rat ankles with adjuvant arthritis. (b) Multinucleated giant cells (arrowheads) at the bone/synovium interface were preferentially stained for β-gal. (c) These multinucleated cells (arrowheads) were positively stained for TRAP, a specific marker for osteoclasts. (d) In vivo expression of Csk protein in rat ankles detected by Western blotting (n = 2 each day). Fold increase in Csk expression normalized by β-actin is shown below each blot. The results were derived from 3 separate experiments, with duplicates performed in each experiment.
Figure 7
Figure 7
Therapeutic effects of Csk virus injection on rat adjuvant arthritis. All rats were immunized with adjuvant in the right foot-pad (day 0). Viruses were intra-articularly injected into ankles on day 7. Data are shown as mean ± SD. (a) Effects of Csk virus injection into right ankles, as evaluated by arthritis score. The arthritis score of the Csk group (n = 10) was significantly lower than that of the WT (n = 10) and LacZ (n = 10) groups on days 21, 28, and 35 (*P < 0.01 Csk vs. LacZ, Csk vs. WT [day 21]; **P < 0.001 Csk vs. LacZ, Csk vs. WT [days 28 and 35]). (b) Effects of Csk virus injection into right ankles, as evaluated by increase in paw volume. The increase in paw volume of the Csk group was significantly less than that of the WT and LacZ groups on days 14, 21, 28, and 35 (*P < 0.05 Csk vs. WT; #P < 0.01 Csk vs. LacZ [day 21]; **P < 0.01 Csk vs. WT, Csk vs. LacZ [day 14]; ##P < 0.001 Csk vs. WT, Csk vs. LacZ [days 28 and 35]). (c) Effects of Csk virus injection into left ankles, as evaluated by arthritis score. The arthritis score of the Csk group (n = 10) was significantly lower than that of the WT (n = 10) and LacZ (n = 10) groups on days 28 and 35 (*P < 0.05 Csk vs. LacZ; #P < 0.05 Csk vs. WT). (d) Effects of Csk virus injection into left ankles, as evaluated by increase in paw volume. The increase in paw volume of the Csk group was significantly less than that of the WT and LacZ groups (*P < 0.05 Csk vs. WT; #P < 0.05 Csk vs. LacZ). (f) On day 35, the right ankles of WT virus–injected rats showed the radiological findings of severe joint destruction — joint space narrowing, erosion, periarticular osteoporosis, and bone spur formation — compared with a normal rat ankle (e). (g) Radiological examination of Csk virus–injected right ankles showing minimal destructive changes. (j) The radiological score of Csk virus–injected right ankles was significantly decreased in comparison with WT and LacZ groups (*P < 0.001 Csk vs. WT, Csk vs. LacZ). (h) Pathohistological findings of WT virus–injected right ankles. Hyperplasia of synovial membrane occupied the articular spaces of talo-tibial (arrowhead a), talo-calcaneal (arrowhead b), and calcaneo-navicular (arrowhead c) joints. (i) Pathohistological findings of Csk virus–injected right ankles. Synovial hyperplasia and invasion into subchondral bone were markedly suppressed. (k) The pathological score of Csk virus–injected right ankles was significantly decreased in comparison with WT and LacZ groups (*P < 0.001 Csk vs. WT, Csk vs. LacZ).
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